IJCRR - 2(9), September, 2010
Pages: 23-28
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DEVELOPMENT OF ULTRAVIOLET SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF DICLOFENAC SODIUM IN STRATUM CORNEUM BY TAPE STRIPPING METHOD
Author: V. A. Arsul, R. Mayee, S. A. Bhale
Category: Healthcare
Abstract:The release of Diclofenac sodium from transdermal gels was studied using the skin stripping method. The tape stripping method in its standardized form is well-suited to determine the dermatopharmacokinetics of topically applied substances. The tape strips contain the amount of corneocytes and the corresponding amount of the penetrated Diclofenac sodium formulation, which was determined by UV spectroscopic method. Simple, rapid, sensitive and accurate UV- Spectrophotometric methods have been developed for estimation of Diclofenac Sodium in stratum corneum. Diclofenac sodium exhibited distinct λmax in methanol at 285nm. Linearity was observed in the concentration range 2-24 \?g/ml. (r2 =0.9845). Amounts of drug estimated from gel formulation applied on skin and recovered through tape stripping method were in good agreement with label claim. The UV spectroscopic method was validated statistically and by recovery studies. The proposed methods are economical and sensitive for the estimation of Diclofenac sodium present in stratum corneum, when applied topically.
Keywords: Diclofenac sodium, Method validation, Recovery study, Tape stripping
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INTRODUCTION
Diclofenac Sodium is chemically Sodium 2-[(2,6-dichlorophenyl)- amino]phenylacetate. It is Non-steroidal anti-inflammatory drug (NSAID) taken to reduce inflammation and as an analgesic reducing pain in conditions such as arthritis or acute injury1 .
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nbutylammonium hydroxide (Cakirer et al., 1999), Spectrofluorimetry using europium (III) as a fluorescent probe (Carreria et al, 1995), Gas and Liquid chromatography in biological fluids (De-Jong et al, 1989). Literature survey revealed the availability of method of estimation of drugs by skin stripping method2 . But a quantitative determination method of diclofenac sodium for studying the bioequivalence of transdermal gel preparations of this drug was not validated. The aim of present investigation is to develop simple, rapid and cost effective analytical method for diclofenac sodium containing gel formulation and its validation.
MATERIALS AND METHOD
Instruments
A Shimadzu UV-Visible Spectrophotometer (UV-1700) with a matched pair of 10 mm quartz cells were used for this experiment.
Materials
Diclofenac sodium was procured as gift sample from Torrent Research Centre, Ahmadabad. The obtained Diclofenac sodium was dissolved in methanol for experimental purpose. The commercially available marketed gel formulation containing Diclofenac sodium, 2% w/w in gel was used for estimation.
Preparation of Standard Stock Solution
The standard stock solution was prepared by dissolving 0.2gm Diclofenac sodium in 200ml methanol to make final concentration of 100μg/ml. Different aliquots were taken from stock solution and diluted with methanol separately to prepare series of concentrations from 2-24 μg/ml. The λ max for Diclofenac sodium in methanol was found to be 285 nm. The calibration curve was prepared by plotting absorbance versus concentration of Diclofenac sodium3 .
Method
The proposed procedure was adopted for determination of Diclofenac sodium in following manner. The Diclofenac diethylamine gel containing 2% diclofenac sodium was applied on the skin. The release of Diclofenac sodium from transdermal gels was studied using the skin stripping method. Tape stripping is a simple and efficient method for the assessment of quality and efficacy of cosmetical and dermatological formulations. The tape stripping method in its standardized form is well-suited to determine the dermatopharmacokinetics of topically applied substances. After topical application and penetration of Diclofenac sodium formulations, the cell layers of the stratum corneum are successively removed from the same skin area using adhesive films4 . The tape strips contain the amount of corneocytes and the corresponding amount of the penetrated Diclofenac sodium formulation, which can be determined by UV spectroscopic method. Shake these tape strips with 60 ml of methanol in a 200-ml volumetric flask and dilute to volume with methanol. From this solution, suitable aliquots were prepared, then these dilutions were scanned in UV region and absorbances were noted at 285 nm and concentration was determined by linear regression equation5 .
RESULTS AND DISCUSSION
The method was validated by studying the following parameters as International Conference on Harmonization guide lines (ICH guide lines 1995) for method validation6.
Linearity
The linearity of the response of the drug was verified at 2 to 100 μg/ml concentrations, but linearity was found to be between 2-24 μg/ml concentrations. The calibration graphs were obtained by plotting the absorbance versus the concentration data and were treated by linear regression analysis. The equation of the calibration curve for Diclofenac sodium obtained y = 0.0048x - 0.0006, the calibration curve was found to be linear in the aforementioned concentrations. The correlation coefficient (r2 ) of determination was R² = 0.9845(Table 1, 2)
Precision
Assay of method precision (intraday precision) was evaluated by carrying out six independent assays of test samples of Diclofenac sodium. The intermediate precision (interday precision) of the method was also evaluated using two different analysts, systems and different days in the same laboratory. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.36, 99.50 and 0.31, 99.60 respectively. (Table 3)
Accuracy (Recovery test)
Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts of the drug in the placebo. The recovery was performed at two levels, 50 and 100% of Diclofenac sodium standard concentration. The recovery samples were prepared in before mentioned procedure. Three samples were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The recovery values for Diclofenac sodium ranged from 99.7 to 101.5%. The result of analysis for the recovery studies were as presented in Tables 4 and 5. The percentage recovery value indicates that there was no interference from the excipients present in the formulation. The developed method is found to be sensitive, accurate, precise and most reproducible.
CONCLUSION
A method for the determination of Diclofenac sodium in the gel formulation which is obtained by tape stripping method has been developed. A good linear relationship (r2 =0.9845) was observed between the concentration ranges of 2-24 μg/mL. The assay of Diclofenac sodium was found to be 99.50%. The high percentage recovery indicates the high accuracy of the method. This demonstrates that the developed spectroscopic method is simple, accurate and reproducible. Thus the developed method can be easily used for the routine quality control of Diclofenac sodium in stratum corneum by tape stripping method.
ACKNOWLEDGEMENTS
The authors’ thanks to Torrent Research Centre, Ahmadabad for providing the diclofenac sodium standard and gel formulation. The authors would like to acknowledge Principal, Dr. Vedprakash patil Pharmacy College, Aurangabad, India for providing facilities for conducting this research.
References:
1. United States Pharmacopoeia, USP 24/NF 19, US Pharmacopoeial Convention Inc2000, USA, pp.2149- 2152.
2. U.V.Sera, M.V.Ramana, In vitro skin absorption and drug release – a comparison of four commercial hydrophilic gel preparations for topical use. The Indian Pharmacist, 2006, 73, 356-360
3. Agrawal YK and Shivramchandra K, Spectrophotometric determination of diclofenac sodium in tablets. J. Pharm. Biomed. Anal. 1991, 9: 97- 100.
4. Knudsen, EA. Isolation of dermatophytes from foot wear with adhesive tape strips. J, of Medical and veterinary Mycology 1986, 25; 59-61
5. Flynn, G.L., Shah, V.P., Tenjarla, S.N., et al., Assessment of value and applications of in vitro testing of topical dermatological drug products. Pharm. Res. 1999, 16, 1325–1330.
6. ICH-Q2R1, Validation of Analytical Procedures: Methodology International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use 1996, Geneva, Switzerland.
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